Abstract GGGGCC hexanucleotide repeat expansions (G4C2), in the non-coding region of the C9orf72 gene on chromosome 9p21, are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. G4C2 expansions in C9orf72 cause a loss of nuclear membrane integrity which in turn leads to mislocalization of nuclear TDP-43 (another ALS causing protein) in the spinal cords of C9 ALS patients. Recently, expanded hexanucleotide repeat binding proteins have been identified in neurons differentiated from induced pluripotent stem cells and motor cortex tissue from C9-ALS patients. Matrin-3 (MATR3) interacts with hexanucleotide repeats in two independent studies. Mutations in MATR3, an RNA binding nuclear matrix protein, are a known genetic cause of ALS. MATR3 interacts with nuclear matrix proteins such as Lamin A suggesting potential role of MATR3 in regulating nuclear matrix architecture and function. MATR3 positive cytoplasmic inclusions have been identified in an ALS patient known to carry the C9orf72 repeat expansion suggesting a potential link between these two forms of ALS. MATR3 binds to the G4C2 in C9orf72 and the binding motif recognized by MATR3 has been found to be enriched in sporadic ALS patients? alternative splicing cassette exons. However, the physiological consequences of the interaction between MATR3 and expanded G4C2 repeats, as well as the relevance of this interaction to ALS pathogenesis, are not yet known. We found that ectopic expression of wild type (WT) MATR3 strongly suppresses neurodegenerative phenotype in a Drosophila model of mutant C9ORF72 hexanucleotide repeat expansions (HNE). We observed that ablating the RNA binding domain in WT-MATR3 blocks its ability to suppress C9-HNE toxicity in vivo, suggesting that the rescue phenotype depends on HNE RNA transcripts binding to WT-MATR3. Here, we propose to investigate this novel interaction between WT-MATR3 and C9ORF72-HNE and its impact on C9ORF72-HNE neurodegenerative phenotype. In this R21 proposal, we will thoroughly examine the relationship between MATR3 and C9 using biochemical, genetic and molecular approaches in mammalian cell cultures, induced pluripotent stem cells from C9 patients and Drosophila models of C9orf72-mediated neurodegeneration.